FOLIA HISTOCHEMICA ET CYTOBIOLOGICA Vol. 42, No. 1, 2004 pp. 41-43

CD3 receptor modulation in Jurkat leukemic cell line

Agnieszka Józ´wik1 , Monika Soroczyn´ska1, Jacek M. Witkowski1 and Ewa Bryl2 1Department of Pathophysiology and 2Department of Immunopathology, Medical University, Gdan´sk, Poland

Abstract: CD3 antigen is a crucial molecule in signal transduction. Although its expression on cell surface is constitutive, dynamic regulation of TCR-CD3 level is probably the most important mechanism allowing T cells to calibrate their response to different levels of stimuli. In our study we examined the role of two main T cell signal transduction pathways in controlling the surface level of CD3 antigen, one based on protein kinase C activity and the other dependent on calcineurin. As an experimental model we used three clones derived from Jurkat cell line, expressing different levels of CD3 antigen surface expression: CD3low (217.6), CD3+(217.9) or CD3low/+ (217.7). The cells were stimulated with PMA or ionomycin, acting directly on PKC and calcineurin, respectively. Prior to the stimulation cells were incubated with PKC inhibitor - chelerythrine or calcineurin blocker - cyclosporine A. Changes in CD3 surface expression were measured by flow cytometry. Only PMA and chelerythrine were able to change CD3 expression suggesting important involvement of PKC in the regulation of its expression. To confirm these findings, PKC activity was estimated in Jurkat clones. Our data demonstrated that Jurkat clones with different CD3 expression showed also different PKC activities, so we conclude that PKC-dependent pathway is the main way of controlling CD3 level on Jurkat clones. Key words: Jurkat cells - CD3 - Protein kinase C

Introduction the principles of such aberrations as well as about the exact mechanism governing the expression of the inves- CD3 antigen is a multimeric protein, consisting of two tigated antigen. Therefore we addressed the question heterodimers: CD3εγ, CD3εδ and one homodimer about the importance of two main T cell signalling CD3ζζ, expressed exclusively on the surface of T cells, pathways, one based on PKC and the other on calci- in combination with TCR chains [10]. Sophisticated and neurin activity, in controlling CD3 surface level. selective mechanisms allow only the expression of com- pletely assembled and functional receptors [7]. A con- stitutive, but at the same time dynamic CD3 expression Materials and methods plays physiologically important role in the regulation of Jurkat human T cell line (E 6.1) heterogeneous in respect to CD3 T cell function [8]. One of the mechanisms responsible surface expression was subcloned, using limited dilutions method, for CD3 down-modulation, occurring physiologically into 3 clones stably expressing different levels of CD3. Clone 217.6 expressed low levels of CD3 (CD3low), clone 217 .9 showed high after T cell stimulation, is dependent on the protein CD3 surface expression (CD3+) and on clone 217.7 CD3 expression kinase C activity. PKC phosphorylates a serine residue was bimodal, low and high (CD3low/+). Cells were cultured in RPMI after recognising a leucine based motif in the CD3γ 1640 (Sigma, USA) with 10% FCS, penicillin, streptomycin and 2 chain, causing internalisation of the whole CD3-TCR mM glutamine (complete medium) at 37˚C in 5% CO2. × 6 complex [2]. As T cell activation is known to correlate Cells were suspended at concentration of 1 10 cells/ml in complete medium and incubated at 37˚C in 5% CO2 for 6 h with with the degree of TCR down-modulation [8], this pro- and without PKC stimulator - PMA and calcineurin stimulator - iono- cess appears to play an important role in the regulation mycin at a concentration of 0.05 µg/ml and 0.5 µM, respectively. Prior of T cell function. Heterogeneity of CD3 surface level to stimulation, the cells were incubated for 1 h in the same is a common feature of many pathologies including T conditions with PKC inhibitor - chelerythrine (0.1 µg/ml) or calcineurin inhibitor - cyclosporine A (1 µM). CD3 surface expression cell lymphoma [11] and still not much is known about was measured by staining with anti-CD3ε monoclonal antibody con- jugated with FITC (DAKO, Denmark). Cytometric analysis was performed on the FACSCalibur (Becton Dickinson) at the De- Correspondence: E. Bryl, Dept. Immunopathology, partment of Clinical Biochemistry, or Galaxy (DAKO) or FAC- Medical University, De˛binki 7, 80-211 Gdan´sk, Poland; Scan (Becton Dickinson) at the Department of Pathophysiology, e-mail: [email protected] 42 A. Józ´wik et al.

Fig. 1. Diversified effect of chelerythrine on CD3 expession depends on the initial receptor level. Jurkat clones express different levels of CD3 antigen: low on 217.6 clone (A), high on 217.9 (B) and ranging from low to high levels on clone 217.7 (C). PMA down-modulated CD3 expression on all clones (D, E, F), but effect of chelerythrine with PMA on CD3 expression was different for clones 217.6 (D) and 217.9 (E). Chelerythrine alone increased CD3 expression on clone 217.6 (G), and on low CD3 expressors of clone 217.7 CD3+ (I). Fig. 2. Varied protein kinase C activity in three Jurkat clones. PKC activity in the Jurkat clones is much higher than in PBMC. The lowest activity was observed in 217.6 clone and high in 217.9 and 217.7 clones. Phosphatidyl serine (PS) increased PKC activity in all investigated cells. PMA decreased PKC activity in 3 clones, while increasing its activity in PBMC. Chelerythrine acted as PKC stimulator for all cells. PBMC treated with the combination of PMA and chelerythrine showed increased PKC activity, in contrast to Jurkat clones.

Medical University of Gdan´sk. Data were analysed with WinM- activator solution (optional). Probes without and with 10 µg of pure DI, version 2.9 (J. Trotter, The Scripps Research Institute). PKC were used as negative and positive controls, respectively. Final PKC activity was examined with the use of PepTag® Assay for reaction mixture of all probes was 25 µl. Cell extracts containing Non-Radioactive Detection of Protein Kinase C (Promega, USA). PKC were added just before incubation which was performed for 30 Cells were initially treated with chelerythrine and later incubated min at 30˚C. Reaction was stopped by boiling in water bath for 10 with/without PMA for 15 min; a portion of cells was left unstimulated min. Probes with 1 µl of 80% glycerol were loaded onto 0.8% agarose as a control of spontaneous PKC expression. Cells were lysed after gel and separated at 100 V for 40 min. Fluorescent bands of different being adjusted to 5×106 in 0.5 ml, in the extraction buffer (25 mM electrophoretic mobility were visualized by UV transillumination Tris-HCL (pH 7.4), 0.5 EDTA, 0.5 EGTA, 0.05% Triton®1 X-100, and the pictures were stored for densitometric analysis using GDS- 10 mM mercaptoethanol, 1 µg/ml leupeptin, 1 µg/ml aprotinin, 0.2 8000 Gel Documentation System and Labworks 4.0 (UVP, UK). mM sodium vanadate, 1% PMSF in 100% ethanol) and then soni- cated for 30 sec using Sonoplus 2070 (Bandelin Electronic, Ger- many). Cell extracts were centrifugated at 10000 × g for 20 min, and Results the membrane and cytoplasmic fraction was collected. The amount of proteins in lysates was measured by Bradford method. One µg of Three Jurkat clones with different CD3 expression were protein was used for further analysis. During PKC assay, PKC used as an experimental model, allowing to investigate activator - phosphatidyl serine (PS), provided by the producer was used alternatively for the enzyme stimulation. Probes prepared in modulation of CD3 surface expression depending on order to estimate PKC activity included 5 µl reaction buffer, 5 µl C1 two signalling pathways in T cells. PMA stimulation peptide, 1 µl peptide protection solution, 9 µl PKC probe, and 5 µl decreased CD3 expression on all Jurkat clones (Fig. CD3 receptor modulation in Jurkat cells 43

1D-F). Neither ionomycin nor calcineurin inhibitor cy- plained by the fact that part of PKC pool in Jurkat clones closporine A exerted any effect on CD3 expression on is initially activated, so PMA initiated its degradation Jurkat cells (data not shown). Chelerythrine exhibited since it is known to exert such effect on active kinase diverse effect on the CD3 antigen expression, dependent form [3]. Chelerythrine considered in some reports as a on its initial level. It increased CD3 surface expression specific PKC inhibitor [5], appeared to act as a stimula- on 217.6 clone when used alone, as well as in combina- tor used at the concentration of 0.1 µg/ml, as suggested tion with PMA, inverting the effect of the stimulator previously [9]. Additionally, it exerted diverse effect on (Fig. 1G). On 217.9 clone (CD3+), chelerythrine not only CD3 expression in the clones depending on the initial did not invert PMA effect, but even increased CD3 level of receptors. Jurkat cells are known to express all down-modulating caused by PMA (Fig. 1E). Effects on isoforms of PKC [1], but as leukemic cells they can have the 217.7 clone, exhibiting CD3 expression ranging some specific isoforms in bigger proportion [4] which from low to high, confirmed previous observations as can be the reason of CD3 initial level-dependent antigen chelerythrine increased the CD3 level in the population regulation mechanism. with low level of the receptor and enhanced the PMA down-modulating effect on the population with high References antigen level (Fig. 1F, I) . These results suggested a very important role of PKC in the regulation of CD3 express- [ 1] Cantrell S, Lucas S, Marais R, Graves JD, Alexander DP (1990) ion. Heterogeneity of protein kinase C expression and regulation in T lymphocytes. FEBS Lett 260: 53-56 To confirm that role, PKC activity was measured. [ 2] Dietrich J, Hou X, Wegener A, Geisler C (1994) CD3γ contains The initial enzyme activity in all Jurkat clones was much a phosphoserine-dependent di-leucine motif involved in down- higher than in normal PBMC (Fig. 2). Different PKC regulation of the T cell receptor. EMBO J 13: 2156-2166 activity was determined in the investigated Jurkat [ 3] Dupuis G, Ahnadi CE, Giguere P, Gravel S, Gagne D, Goulet clones. Clone 217.6 exhibited the lowest PKC activity, AC (2000) Chronic PMA treatment of Jurkat T lymphocytes results in decreased protein tyrosine phosphorylation and inhibi- while clones 217.9 and 217.7 showed similar enzyme tion of CD3 but not Ti dependent antibody triggered Ca2+ activity. Phosphatydyl serine increased PKC activity in signaling. J Leukoc Biol 68: 293-300 all three Jurkat clones as well as in PBMC. Surprisingly, [ 4] Filelds AP, Murray NR (1997) Atypical protein kinase C iota PMA appeared to inhibit PKC activity in Jurkat clones, protects human cells against drug-induced . but still was acting as an activator in PBMC. Paradoxi- J Biol Chem 272: 27521-27524 [ 5] Herbert JM, Augerean JM, Gleye J (1990) Chelerythrine is a cally, chelerythrine increased PKC activity in all inves- potent and specific inhibitor of protein kinase C. Biochem tigated cell types. In combination with PMA it decreased Biophys Res Commun 172: 993-999 PKC expression on clone 217.6 to a much greater extent [ 6] Jarza˛bek K, Laudan´ski P, Dzie˛cioł, Dobrowolski M, Wolczy- than on two other Jurkat clones (Fig. 2). n´ski S (2002) Protein kinase C involvement in proliferation and survival of breast cancer cells. Folia Histochem Cytobiol 40: 193-194 Discussion [ 7] Klausner RD, Letourneur F (1992) A novel di-leucine motif and a tyrosine based motif mediate lysosomal targeting and endocy- Our experimental model based on three Jurkat clones tosis of CD3 chains. Cell 69: 1143-1157 with different CD3 expression, allowed us to examine [ 8] Lanzavecchia A, Viola A (1996) T cell activation determined by T cell receptor number and tunable thresholds. Science 273: whether signalling pathway dependent on protein kinase 104-106 C and the one dependent on calcineurin, have any in- [ 9] Pezzuto JM, Lee SK, Woongchon M, Luyengi L, Mehta RG, fluence on CD3 expression regulation, in relation to its Kawanishii K, Fong HS, Beecher WW, Kinghorn AD (1998) initial level of expression. Results obtained from stimu- Angoline and chelerythrine, benzophenanthridine alkaloids that lation and inhibition experiments measuring CD3 ex- do not inhibit protein kinase C. J Biol Chem 272: 19829-19833 [10] Punt JA, Roberts JL, Kearse KP, Singer A (1994) Stechiometry pression by flow cytometry suggested that calcineurin of the T cell antigen receptor (TCR) complex: each TCR/CD3 does not play a significant role in CD3 modulation, while complex contains one TCR alfa, one TCR beta, and two CD3 PKC is a crucial enzyme in this process. PKC has been epsilon chains. J Exp Med 180: 587-593 previously reported to participate in the proliferation and [11] Sikora J, Z˙ eromski J (2002) Expression of TCR-zeta chain and survival of cancer cells [6]. We showed that in all Jurkat apoptosis in subpopulations of tumor associated lymphocytes (TALs) from malignant pleural effusions. Folia Histochem Cy- clones PKC activity was much higher than in PBMC tobiol 40: 347-351 from healthy donors. Additionally, Jurkat cells reacted differently than normal lymphocytes to PMA treatment, Accepted July 23, 2003 by lowering PKC activity. This difference can be ex-